CN102460174B - One group of novel low abundance human plasma protein biomarker is used to carry out clinical diagnosis to liver fibrosis - Google Patents

One group of novel low abundance human plasma protein biomarker is used to carry out clinical diagnosis to liver fibrosis Download PDF

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CN102460174B
CN102460174B CN201080031411.7A CN201080031411A CN102460174B CN 102460174 B CN102460174 B CN 102460174B CN 201080031411 A CN201080031411 A CN 201080031411A CN 102460174 B CN102460174 B CN 102460174B
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antibody
albumen
biomarker
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polypeptide
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CN102460174A (en
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雷蒙德·A·德韦克
贝文·甘加达兰
妮科尔·齐兹曼
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Danuo Oxford Science Co ltd
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University of Oxford
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Abstract

The present invention proposes one group of novel human plasma protein's biomarker for diagnosing liver fibrosis and cirrhosis.Do not assess the non-invasive manner of liver fibrosis at present reliably.Based on the protein science research of 2D-PAGE for differentiating potential fiberization biomarker.Analyze the blood plasma of the patient from the cirrhosis suffering from hepatitis C virus (HCV) infection-induced.Authenticated relevant to liver scarring and potential with virus infections relevant several protein.These albumen comprise 14-3-3 albumen ζ/δ, adiponectin, first albumin, α-1-antitrypsin, α-2-HS-glycoprotein, apoC-III, apo E, C4b-associated proteins β chain, the Complement C_3 dg of complete/cracking, corticosteroid-binding globulin, fibrinogen γ chain, pH? 5.46 to pH? the β hoptoglobin at 5.49 places, hoptoglobin associated protein, hemopexin, immunoglobulin (Ig) J chain, rich leucine α-2-glycoprotein, fat transfer inhibitor albumen, retinol-binding proteins, Serum paraoxonase/Arylesterase activity 1, sex hormone binding globulin and zinc-α-2-glycoprotein.These biomarkers can with the polypeptide conbined usage in international publication WO/2008/031051.The concentration of these new biomarker can use immunoassay to measure, and wherein, described concentration will reflect Fibrotic degree.The present invention proposes the Fibrosis score classification of each in described new biomarker.The result that the score value of all new biomarker is added will provide more reliable fibrosis index, but not detect single biomarker.

Description

One group of novel low abundance human plasma protein biomarker is used to carry out clinical diagnosis to liver fibrosis
The cross reference of related application
This application claims the U.S. Provisional Application the 61/178th submitted on May 14th, 2009, the right of priority of No. 334.
Technical field
The application relates generally to the method for use one group of antibody diagnosis liver fibrosis or cirrhosis, described antibody target one group of abundance low-down Novel liver scarring biomarker.These novel proteins also can be used as hepatitis, the biomarker of hepatocellular carcinoma (HCC) and the drug targets of liver scarring, hepatitis and HCC.
Background technology
Liver fibrosis: liver fibrosis is wound healing response, this wound healing response with the over-agglomerate of cicatricial tissue in liver (i.e. extracellular matrix) for feature.The normal configuration key element of tissue is replaced by excessive non-functional cicatricial tissue.Puncture liver carries out biopsy to be diagnosis and to assess Fibrotic Main Means, but has ample evidence to show this technology to have many restrictions and shortcoming, comprises the death in patient's discomfort, pain, hemorrhage and only a few case.In addition, if fiberization is not homogeneous in the middle of whole liver, so, biopsy may be unreliable.Liver fibrosis can be caused by the many factors comprising alcohol and virus.
Cirrhosis: cirrhosis is the most serious form of liver scarring, and, different from liver fibrosis, it has been generally acknowledged that cirrhosis is irreversible and has tubercle.In Britain, cirrhosis causes the death more than 6000 examples every year, in the U.S., cirrhosis causes about 27 every year, the death of 000 example, and cirrhosis is the ninth-largest lethal factor (MacSween etc., (2002), PathologyoftheLiver, the 4th edition, ChurchillLivingstone).Cirrhosis is the major risk factors of HCC, and in this stage of liver cancer, unique treatment means is liver transplant.In viral-induced liver cancer case, liver scarring and HCC can recur after the transfer.Be necessary at reversible liver scarring commitment diagnosis of fibrosis, thus irreversible cirrhosis can be prevented.
Hepatitis C virus: the whole world about 1.7 hundred million population (namely world population 3% (see, such as WHO, J.Viral.Hepat.1999; 6:35-47)) infect hepatitis C virus (HCV, HepC), the U.S. about four a population of one million infects hepatitis C virus.HCV is the Etiological of liver fibrosis and cirrhosis.The individuality of about 80% acute infection HCV becomes chronic infection.Therefore, HCV is the Etiological of chronic hepatitis.Once this virus of chronic infection, almost never remove if do not carry out treatment virus.In only a few case, HCV infection causes Clinical Acute disease even hepatic failure.Chronic HCV infection can be significantly different between individuality, some of them individuality has unconspicuous liver diseases or have first degree liver diseases and never complication occur clinically, and other individuality has clinically significantly chronic hepatitis continue to develop into liver fibrosis and cirrhosis.About 20% in the individuality of HCV that carry developing into cirrhosis will develop into end-stage liver disease and the risk increase developing into primary carcinoma of liver.
The method improved is needed to diagnose liver fibrosis in patient and cirrhosis.
Summary of the invention
The invention provides the method for detection fibers and cirrhosis.
In one embodiment, the invention provides the method for detection fibers and cirrhosis, described method comprises: (a) measures the level of relevant (HF-ASSOCIATED) polypeptide of the HF-taken from the biological specimen of patient; And the control level of described level (a) with described HF-related polypeptide compares to determine described Fibrotic positive diagnosis or negative diagnostic by (b).These biomarkers can be applicable to show Fibrotic any disease, the such as liver fibrosis of described fiberization, kidney fibrosis, cardiac fibrosis (cardialfibrosis), fibrosis of skin, pancreatic fibrosis etc., but described in specific embodiment, fiber turns to liver fibrosis.The present invention selects the polypeptide in lower group: 14-3-3 albumen ζ/δ, adiponectin, first albumin, α-1-antitrypsin, α-2-HS-glycoprotein, apoC-III, apo E, C4b-associated proteins β chain, the Complement C_3 dg of complete/cracking, corticosteroid-binding globulin, fibrinogen γ chain, the β hoptoglobin at pH5.46 to pH5.49 place, hoptoglobin associated protein, hemopexin, immunoglobulin (Ig) J chain, rich leucine α-2-glycoprotein, fat transfer inhibitor albumen, retinol-binding proteins, Serum paraoxonase/Arylesterase activity 1, sex hormone binding globulin and zinc-α-2-glycoprotein.These biomarkers can with the polypeptide conbined usage in international publication WO/2008/031051, and described polypeptide comprises inter-α-trypsin inhibitor heavy chain H4 fragment, α 1 antichymotrypsin, Apolipoprotein L1, prelbumin, albumin, CD5 antigen sample albumen isoform, beta 2 glycoprotein I, alpha2 Macroglobulin and immunoglobulin (Ig) composition, α 1, the α 2 of hoptoglobin and β chain, complement component (C3, C4 and H factor-associated protein 1), adiponectin, ApoE, factor, CLU and proangiotensin.In other embodiments, fiberization comprises the difference regulation and control of HF-related polypeptide.In other embodiments, sample takes from serum or blood plasma.
In another embodiment, the invention provides the method detecting HF-related polypeptide, described method comprises: a) from separating bio sample the patient suffering from fiberization or cirrhosis, b) separating bio sample in Fibrotic patient is never suffered from, c) analyze from sample a) and b) with 2D-PAGE, and d) compare 2D-PAGE result to differentiate the polypeptide of differential expression between the patient suffering from and do not suffer from fiberization or cirrhosis.When using 2D-PAGE, biomarker can use conventional wide pH range detection, described wide pH scope such as pH3 to pH10, pH3 to pH11 or pH4 to pH7.The low-down biomarker of abundance can detect with any narrow pH range of pH3 to pH11, and described narrow pH range is such as but not limited to pH3 to pH5.6, pH3 to pH6, pH3.9 to pH5.1, pH4.7 to pH5.9, pH5 to pH8, pH5.3 to pH6.5, pH5.5 to pH6.7, pH6 to pH11, pH6.2 to pH7.5, pH6.3 to pH8.3, pH7 to pH10, pH7 to pH11, pH3.5 to pH4.5, pH3 to pH7 and pH6 to pH9.The 2D-PAGE gel covering these narrow pH ranges also can be used for differentiating the new biomarker in other diseases and drug targets.
In another embodiment, the invention provides the method for fiberization classification of severity, described method comprises: a) measure the level taking from least one HF-related polypeptide in the biological specimen of patient; And b) by the described HF-related polypeptide level in the biological specimen of described patient and comparing without the level of fiberization to the described HF-related polypeptide in the patients of cirrhosis of measuring in advance.
In another embodiment, the invention provides kit useful in Fibrotic prognosis in untreated individuality and over the course for the treatment of, described kit comprises HF-related agents, wherein said medicament specific detection HF-related polypeptide.In specific embodiment, described medicament is antibody in conjunction with HF-related polypeptide or its function equivalent.These antibody can be used for carrying out immunoassays, such as enzyme linked immunosorbent assay (ELISA) (ELISA), radioimmunoassay, protein spots trace, Western blot (Westernblot), turbidimetry, scattered light urbidmetry etc.HF-related polypeptide also can use non-antibody approach to come quantitatively, carries out multiple-reaction monitoring such as, but not limited to, use mass spectrum.Described kit can comprise at least one target further, and described target specificity ground detects another gene or gene outcome of being used as prognostic indicator.
In another embodiment, the invention provides the method measuring Fibrotic prognosis, described method comprises: (a) measures the level of the HF-related polypeptide taken from the biological specimen of patient; And the control level of the described level of (a) and described HF-related polypeptide compares to determine described Fibrotic positive diagnosis or negative diagnostic by (b).
Accompanying drawing explanation
This few width figure of Fig. 1 to Fig. 4 is presented at the change observed in the expression of main new biomarker.Every width figure shows the magnification region of 2D-PAGE gel, and wherein, the relevant position of the albumen differentiated is circled.The representative plasmagel image of healthy individuals and liver cirrhosis patient is shown in figure.
Fig. 1 shows fat transfer inhibitor albumen and occurs in normal plasma but reduce in liver cirrhosis patient blood plasma.
Fig. 2 shows zinc-α-2-glycoprotein and occurs in normal plasma but reduce in liver cirrhosis patient blood plasma.
The unique point (feature) that Fig. 3 shows the β hoptoglobin at pH5.46 to pH5.49 place reduces.Top figure-β hoptoglobin spot be uniformly distributed array, this array display normal plasma and liver cirrhosis patient blood plasma without marked difference.The enlarged image of the β hoptoglobin spot of the figure-observe at about pH5.46 to pH5.49 place of below, demonstrates the β hoptoglobin spot observed at about pH5.46 to pH5.49 place and occurs in normal plasma but reduce in liver cirrhosis patient blood plasma in figure.
The cracking that Fig. 4 shows Complement C_3 in cirrhosis reduces.Figure-display Complement C_3 the dg of top does not occur but occurs in liver cirrhosis patient blood plasma in normal plasma.The fragment of the Complement C_3 α chain of the figure-be presented at before the thioesters site of Complement C_3 α chain of below occurs but does not occur in liver cirrhosis patient blood plasma in normal plasma.
Embodiment
The following invention describing summary and sum up above.But, the invention is not restricted to described particular methodology, testing program, clone, animal species, construct, reagent and can changing in itself.Similarly, term used herein only describes particular implementation, is not intended to limit scope of the present invention.
When compared with healthy individuals, inventor of the present invention finds differential expression in the human plasma sample of the liver cirrhosis patient that multiple protein is brought out at HCV-.This discovery is compared these serum samples with the technology providing discrete protein spot by being used in two-way protein isolate in gel-type vehicle and realizing.The method uses the close limit pH value Stationary pH gradient gel of pH3 to pH5.6, and this pH gradient gel is different from the Stationary pH gradient gel of the pH3 to pH10 used in WO/2008/031051.
Unless otherwise defined, all technical terms used herein are identical with the generally understanding of person of ordinary skill in the relevant with the implication of scientific terminology.
With regard to description and disclosed object, all public publications mentioned herein and patent are incorporated to herein by reference, such as, and the construct described in public publication that can be combined with content of the present invention and methodology.Public publication discussed above and full content thereof were provided for separately open before the applying date of the present invention.Be not interpreted as herein admitting because prior to this disclosing of the present invention, the present inventor does not enjoy in first right.
A. define
For simplicity, the implication of some terms and the phrase adopted in instructions, embodiment and additional claim is provided below.
Unless the context clearly dictates otherwise, singulative (" a " " an " and " the ") comprises plural reference.
" 2D-PAGE " refers to two-dimensional polyacrylamide gel electrophoresis.
" ELISA " refers to enzyme linked immunosorbent assay (ELISA).
" HCC " refers to hepatocellular carcinoma.
" HCV " refers to hepatitis C virus.
" HF " refers to liver fibrosis.
" kDa " refers to kilodalton.
" PBS " refers to phosphate buffered saline (PBS).
" PBS-T " refers to the PBS solution containing tween.
" biological specimen " comprise take from organic, can be used for diagnosing or the multiple sample type of monitoring analysis.This term comprises other liquid samples, solid tissue's sample (as biopsy sample) of blood and biogenetic derivation, or resultant tissue culture or cell and offspring thereof.In addition, this term can comprise circulating tumor cell or other cells.This term especially comprises clinical sample, and comprises cell, cell conditioned medium liquid, cell pyrolysis liquid, serum, blood plasma, urine, amniotic fluid, biofluid and the tissue samples in cell chulture further.The sample that this term processes after also comprising acquisition by any way, described processing example is as by agent treated, dissolving, some component of enrichment.
" biomolecular sequence " or " sequence " refers to all or part of of polynucleotide or peptide sequence.
" BLAST " refers to basic Local Alignment Search Tool (BasicLocalAlignmentSearchTool), and it is the technology detecting the nonseptate subsequence mated with given search sequence." BLASTP " is blast program amino acid query sequence and protein sequence databank compared." BLASTX " is the blast program six frame conceptual translation products and the protein sequence databank of nucleotide query sequence (double-strand) compared.
" cancer ", " neoplasm " and " tumour " that be used interchangeably in this article refers to the cell or tissue showing aberrant growth phenotype, and described aberrant growth phenotype is significantly out of control for feature with cell proliferation.Method and composition of the present invention be especially applied to before (that is, optimum) before cancer, pernicious, transfer, transfer with the cell of non-diverting.
" fiberization is regulated to feature with the difference of HF-related polypeptide " refers to the patient with the tissue showing scarring, and HF-associated protein has differential expression in the tissue of described scarring.
" fibrosis phenotype " refers to the multiple biological phenomenon being characterized as brotic cells.This phenomenon can change with Fibrotic type, but fibrosis phenotype usually formed by cicatricial tissue in abnormal conditions differentiate.
" cell type " refers to cell from given source (e.g., tissue or organ) or the cell that is in given differentiation state, or the cell relevant to given pathology or genomic constitution.
" complementary " refer to the topological compatibility of probe molecule and its target interactive surfaces or mutually match.Target and its probe can be described as complementary, and the feature of surface in contact is complimentary to one another.
Term " detectable " refers to the expression of polypeptides pattern observable with technology well known to those skilled in the art using and describe in the application.Such as, expression of polypeptides comes " detection " by standard technique (comprising the immunoassays of such as Western blot and so on).
" diagnosis (" diagnosis " and " diagnosing ") " generally include determine patient to the neurological susceptibility of disease or imbalance, determine whether patient suffers from disease or imbalance at present, carries out prognosis (such as to the patient suffering from disease or imbalance, to shifting the cancer state of front or transfer or Fibrotic discriminating) and diagnostic evaluation (therametrics) (such as, monitoring the symptom of patient to provide the information about result for the treatment of or effect).
" differential expression " refers to the difference of difference and the matter measured in the temporal expression patterns and tissue expression pattern of gene.Such as, the gene of differential expression can make it express to be activated or not activate completely in normal condition and morbid state.This gene regulated and controled in nature can show expression pattern in given tissue, cell type or the serum/plasma of patient, described expression pattern is detectable under contrast state or morbid state, or all can detect under contrast state and morbid state but have different expression.As used herein " albumen of differential expression " refers to the amino acid sequence of the albumen having identified differential expression uniquely, thus the detection of the albumen of differential expression in sample is associated with the appearance of the albumen of differential expression in sample.
" expression " typically refers to polynucleotide sequence experience and successfully transcribes and translate, thus expression can the amino acid sequence of detection level or the process of albumen.In some linguistic context, express the generation referring to mRNA.In other linguistic context, expression is the generation of finger protein or its fragment.Described fragment is cut by enzyme or normal condition or the distinctive bioprocess of morbid state produce.
" expression product " or " gene outcome " be when the gene in organism is transcribed translation or posttranslational modification time the biomolecule that produces, such as albumen or mRNA.
" protein fragments " is a part for finger protein.Such as, protein fragments can comprise the polypeptide obtained from the full-length proteins of cultured cells separation by digestion.In one embodiment, protein fragments comprises at least about 6 amino acid.In another embodiment, described fragment comprises at least about 10 amino acid.In yet, protein fragments comprises at least about 16 amino acid.
In the context of this application, term " function equivalent " refers to have and all or part of natural functional characteristic of HF-associated protein basic simlarity or the albumen of architectural characteristic.Term " function equivalent " is intended to comprise " fragment ", " muton ", " derivant " of natural HF-associated protein, " allele ", " crossbred ", " variant ", " analog " or " chemical derivative ".
In the context of immunoglobulin (Ig), term " function equivalent " refers to the immunoglobulin molecules shown with the Immunological binding properties of maternal immunoglobulin basic simlarity." Immunological binding properties " refers to the noncovalent interaction type occurred between immunoglobulin molecules and the specific antigen of this immunoglobulin (Ig).In fact, such as, the function equivalent of monoclonal antibody immunity globulin can suppress parent monoclonal antibody to be combined with its antigen.Function equivalent can comprise F (ab ') 2 fragment, F (ab) molecule, Fv fragment, display technique of bacteriophage single chain variable fragment (scFv), single domain antibody, chimeric antibody etc., as long as immunoglobulin (Ig) shows the characteristic of maternal immunoglobulin.
Term " fusion " refers to the albumen be made up of two or more polypeptide, although typically, described polypeptide does not connect with native state, and described polypeptide is connected to form single continuous polypeptide by its corresponding amino and carboxyl terminal by peptide bond.Should be understood that, two or more polypeptide moiety directly can be connected or be indirectly connected by peptide connector/spacer region.
" gene " refers to the polynucleotide sequence comprising and produce polypeptide or the regulating and controlling sequence needed for precursor and coded sequence.Polypeptide can be encoded by complete encoding sequence or is encoded by any part of coded sequence.Gene can form continual coded sequence or gene one or more intrones that can to comprise with suitable splice site be border.In addition, gene can comprise one or more and modify in code area or non-translational region, and described modification can affect biologically active or chemical constitution, expression speed or the expression regulation mode of expression product.Such modification includes but not limited to: the sudden change of one or more nucleotide, insertion, disappearance and replacement.On this point, the gene modified like this can be described as " variant " of " natural " gene.
" gene expression " refers to that polynucleotide sequence experience is successfully transcribed and translates, thus express can the process of nucleotide sequence of detection level.
As used herein term " homology " refers to complementary degree.Homeologous or complete homology (that is, consistent) can be there is.Partial complementarity sequence is the sequence of the hybridization suppressing identical sequence and target polynucleotide at least partly; Partial complementarity sequence also refers to using function term " basic homology ".Under low stringency condition, hybridization assays (southern blotting technique (Southernblot) or RNA trace (Northernblot), solution hybridization etc.) can be used to detect the suppression that fully-complementary sequence and target sequence are hybridized.Under low stringency condition, the sequence of basic homology or probe will be competed and suppressed the combination of the sequence of complete homology or probe and target sequence (that is, hybridizing).This is not say that low stringency condition is the condition allowing non-specific binding; Low stringency condition requires that being bonded to each other of two sequences interacts specific (that is, optionally).Do not have non-specific binding by using the second target sequence to check, described second target sequence even lacks the partial complementarity degree consistance of 30% (e.g., lower than); When not having non-specific binding, probe can not be hybridized with the second non-complementary target sequence.
" individuality ", " patient ", " host " and " patient " that be used interchangeably herein refers to any mammal patient needing to diagnose, process or treat.In a preferred embodiment, individuality, patient, host or patient are the mankind.Other patients comprise the known marmot and the duck that there occurs HCC and hepatitis.Other patients can include, but are not limited to: ox, horse, dog, cat, cavy, rabbit, rat, primate and mouse.
" separation " refers to polynucleotide, polypeptide, immunoglobulin (Ig) or host cell in the environment different from the environment of natural generation polynucleotide, polypeptide, immunoglobulin (Ig) or host cell.
" mark " refers to directly can be provided the medicament of detectable signal or provide the medicament of detectable signal by interacting with one or more additional members in signal generation system.Can direct-detection and the mark that can use in the present invention comprises fluorescence labeling.Concrete fluorophor comprises fluorescein, rhodamine, BODIPY, cyanine dye etc.The present invention also considers to use radioactive isotope (such as 35s, 32p, 3h, etc.) as mark.Also the colourity of such as collaurum or coloured glass or plastics (as polystyrene, tygon, latex) pearl and so on can be used to mark.See, such as, United States Patent (USP) the 4th, 366, No. 241, the 4th, 277, No. 437, the 4th, 275, No. 149, the 3rd, 996, No. 345, the 3rd, 939, No. 350, the 3rd, 850, No. 752 and the 3rd, 817, No. 837.
Term " normal physiological conditions " refers to lived organism or intracellular representative condition.Although some organs or organism provide extreme condition, but in organism and intracellular environment usually at about pH7 (namely, from pH6.5 to pH7.5) change, comprise water as primary solvent, and to be present in higher than 0 DEG C and lower than under the temperature conditions of 50 DEG C.Different salinity depends on organ for referencial use, organism, cell or cellular compartment.
" polynucleotide " and " nucleic acid " that are used interchangeably herein refer to the polymerized form of the nucleotide (ribonucleotide or deoxyribonucleotide) of any length.Therefore, these terms include, but are not limited to: DNA or RNA of strand, double-strand or multichain, genomic DNA, cDNA, DNA RNA hybrid or comprise purine bases and pyrimidine bases or other natural, chemistry or biochemical modification, the polymer of non-natural or derivative nucleotide base.These terms also include, but are not limited to: mRNA or cDNA comprising intron sequences.The skeleton of polynucleotide can comprise sugar and phosphate group (as usually found in RNA or DNA) or modify or the sugar of replacement or phosphate group.Alternatively, polynucleotide skeleton can comprise the polymer of the synthesis subunit of such as phosphoramidite and so on, and therefore polynucleotide skeleton can be the phosphoramidate of oligodeoxynucleotide or the phosphoramidate-phosphate diester oligomer of mixing.Polynucleotide can comprise nucleotide (such as methylated nucleotide and nucleotide analog), uracil, other sugared and such as fluororibose and monothioester and so on linking groups of modification, and nucleotide side chain.Nucleotide sequence can be interrupted by non-nucleotide components.Polynucleotide can be modified after polymerisation further, such as, modify by being combined with marked member.This define being modified to of the other types comprised add cap, replace in the nucleotide of natural generation with analog one or more, and introduce the method that polynucleotide are connected to albumen, metallic ion, marked member, other polynucleotide or solid support.Term " polynucleotide " also comprises peptide nucleic acid.Polynucleotide can comprise genomic DNA, cDNA or DNA RNA hybrid further.
" polypeptide " and " albumen " that be used interchangeably herein refers to the amino acid whose polymerized form of any length, can comprise translation, untranslated, chemical modification, biochemical modification and derivative amino acid.Polypeptide or albumen can be natural generation, restructuring or synthesis or these any combination.In addition, polypeptide or albumen can comprise the albumen of natural generation or the fragment of peptide.Polypeptide or albumen can be individual molecule or can be multi-molecular complex.In addition, such polypeptide or albumen can have the peptide backbone of modification.These terms comprise fusion, and described fusion comprises the fusion containing heterologous amino acid sequence, containing allos and homologous leader sequences, containing or fusion containing N-terminal Methionine residue, immune labeled albumen etc.
" physique of easily catching an illness " of disease or imbalance refers to the individual neurological susceptibility to this disease or imbalance.Such as, compared with normal/wild type individuality, the individuality of susceptible more may suffer from cancer or fiberization statistically.
Term " prognosis (" prognosis " and " prognose ") " refers to behavior or the technology of predictive disease process.In addition, this term refer to by desired by the general process of disease or surviving from disease and the prospect recovered shown by the special characteristic of individual case.In addition, this term refers to behavior according to its S&S identifying disease or technology.
Term " prognostic indicator " or " index " refer to can be used as prognosis according to or basis anything to prognosis according to or relevant anything in basis.These terms refer to any basis or the basis of antidiastole further, comprise the characteristic of test result as described herein and gene expression, and disease or illness are distinguished mutually with the other diseases or illness showing similar symptom.In addition, term " index " or " prognostic indicator " refer to any basis or the basis of the characteristic comprising test result as described herein and gene expression, and term " index " or " prognostic indicator " can be used for distinguishing the possible process of malignant disease.
" albumen trapping agent " refers to can by protein combination in self molecule or multi-molecular complex.In one embodiment, albumen trapping agent is with the binding partners of substantially special mode in conjunction with them.In one embodiment, albumen trapping agent can show the dissociation constant (KD) lower than about 10-6.Albumen trapping agent can comprise the biomolecule of such as albumen or polynucleotide and so on.This biomolecule can comprise further natural generation, restructuring or synthesis biomolecule.The example of albumen trapping agent comprises immunoglobulin (Ig), antigen, acceptor or other albumen or its part or fragment.In addition, should be understood that, albumen trapping agent is not limited to by means of only noncovalent interaction and the interactional medicament of its binding partners.On the contrary, albumen trapping agent also can be covalently attached to the albumen combined with them.Such as, albumen trapping agent after bonding can with its binding partners generation photo-crosslinking.
" sequence identity " refers to similar or complementary degree.Partial identity or crash consistency can be there is.Partial complementarity sequence is the sequence suppressing identical sequence and target polynucleotide to be hybridized at least partly; Also using function term " basically identical " is referred to.Under low stringency condition, hybridization assays (southern blotting technique or RNA trace, solution hybridization etc.) can be used to detect the suppression of the hybridization of fully-complementary sequence and target sequence.Under low stringency condition, basically identical sequence or probe will be competed and suppressed the combination of completely the same sequence or probe and target sequence (that is, hybridizing).This is not say that low stringency condition is the condition allowing non-specific binding; Low stringency condition requires that the combination each other of two sequences interacts specific (that is, optionally).Do not have non-specific binding can use the second target sequence to check, described second target sequence even lack partial extent complementarity (as, consistance lower than 30%), when not having non-specific binding, probe can not be hybridized with the second incomplementarity target sequence.
Herein two nucleic acid or the conforming other method of peptide sequence observation sequence are comprised with reference to the identical residue in two sequences when carrying out the comparison of maximum correspondence in specific region.The percentage of as used herein sequence identity refers to the value measured by comparing two best alignment sequences in comparison window, wherein compared with the reference sequences (do not comprise and add or lack) of the best comparison of two sequences, in comparison window, part polynucleotide sequence can comprise interpolation or disappearance (that is, breach).Described percentage is calculated by following manner: the number being determined at the identical nucleic acid base position all occurred in two sequences is counted out to produce match bit, count out divided by the sum in site in comparison window by match bit, then result is multiplied by 100, thus produces the percentage of sequence identity.
" stringent condition " refer to probe with this understanding can hybridize with its target polynucleotide sequence and not with the condition of other sequence hybridizations.Stringent condition is sequence-dependent (such as, longer sequence specific hybrid under the condition of higher temperature).Usually, select stringent condition to be about 5 DEG C, lower than the heat fusion joint (Tm) of particular sequence under the ionic strength limited and pH condition.50% temperature (under the ionic strength limited, pH and polynucleotide concentration conditions) of hybridizing with probe and the target sequence of target complement sequence when Tm is balance.Typically, stringent condition to short probe (such as, 10 to 50 nucleotide) be about pH7.0 to about pH8.3, salinity is at least about the Na ion concentration (or other salt) of 0.01M to about 1.0M, and temperature is at least about the condition of 30 DEG C.Stringent condition also realizes by adding the destabilizing agent of such as formamide and so on.
" basic purifying " refers to the compound separated from physical environment, and described compound is at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 83%, at least about 85%, at least about 88%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%, or at least about 99.99% or more not containing other component of combination natural with it.
" target protein " refers to the polypeptide with albumen trapping agent specific hybrid or combination, and described polypeptide derives from biological specimen usually.The existence of described target protein or shortage can be detected, or described target protein can by quantitatively.Described target protein has can the structure of corresponding albumen trapping agent identification of directed target.Described target protein or amino acid also can more large protein pointed by finger protein trapping agent specific substructure or expect the one-piece construction (such as, gene or mRNA) detecting its expression.
Term " treatment (" treatment ", " treating ", " treat ", etc.) " refer to the pharmacology and/or physiologic effect that obtain expection.Described effect can be preventative with regard to prevent disease wholly or in part or its symptom, and/or described effect is just partially or completely stable or partially or completely can be with regard to cure diseases and/or the adverse effect that caused by disease curative." treatment " comprises any treatment of mammal (especially the mankind) disease, and comprise: there is disease or symptom in (a) prevention, described patient can be easy to infect described disease or symptom but not yet be diagnosed as this disease or symptom in patient; B () suppresses disease symptoms, that is, stop it to develop; Or (c) alleviate disease symptoms, that is, make disease or symptom restore.
B.HF-related polypeptide
On the one hand, the present invention relates to " HF-related polypeptide ", described " HF-related polypeptide " comprises its differential expression polypeptide relevant to liver fibrosis.HF-related polypeptide also comprises the variant of the albumen of natural generation, wherein such variant or basic simlarity identical with the albumen of natural generation.Generally, when being detected by BLAST, the sequence of variant polypeptide and HF-related polypeptide described herein have the sequence identity at least about 80%, usually have the sequence identity at least about 90%, and more generally, have the sequence identity at least about 98%.Variant polypeptide can be natural or non-native glycosylation.
Generally, as with method well known in the art (such as BLAST) measure, the variant of HF-related polypeptide described herein has and is greater than at least about 65%, maybe can be greater than sequence identity at least about 99.99% at least about 70%, at least about 75%, at least about 80%, at least about 83%, at least about 85%, at least about 88%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 99.9%.
In one embodiment, variant HF-related polypeptide can be mutant polypeptide.Sudden change in HF-related polypeptide can be produced by 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor, interpolation or disappearance (but being not limited thereto).49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor can be conserved amino acid and replaces or remove nonessential amino acid whose replacement.Generally, conserved amino acid replaces is the 49-Phe ,82-Ser,115-Arg,144-Met,145-Asn ,161-Arg,169-Met Human Connective tissue growth factor keeping being substituted amino acid whose conventional charge, hydrophobicity, water wettability and/or steric effect.
In the HF-related polypeptide of some sudden changes, amino acid can be substituted to change phosphorylation site or acetylation sites.
Importantly, variant polypeptide can be designed to keep or have the biologically active of the albumen specific region (such as, Functional domains and/or show the region of pass together in order, wherein polypeptide is protein family member) of raising.For generation of the amino acid change of variant selection can based on the thermal stability of amino acid whose accessibility (inner with outside), variant polypeptide, expect glycosylation site, expect that the expection in disulphide bridges, the metal-binding sites of expection and proline ring replaces.Halfcystine exhausts that mutain can according to United States Patent (USP) the 4th, the disclosure of 959, No. 314 and producing.
Variant also comprises the fragment of HF-related polypeptide disclosed herein, especially bioactive fragment and the fragment corresponding to functional domain.Typically, target fragment will be at least about 10aa to long at least about 15aa, usually long at least about 50aa, and be 300aa or longer.Protein variant described herein is by the polynucleotide encoding in the scope of the invention.
There is provided in the environment that HF-related polypeptide of the present invention generates at non-natural, such as, be separated from the environment of its natural generation.In some embodiments, HF-associated protein exists with the form of basic purifying.
C.HF-related agents: correctives and binding partners
On the other hand, the invention provides " HF-related agents ", described HF-related agents refers to the molecule be made up of " HF-related polypeptide binding partners ".
HF-related polypeptide binding partners is the molecule being incorporated into HF-related polypeptide.Exemplary polypeptide binding partners is immunoglobulin (Ig).The biologically active of the adjustable HF-related polypeptide of binding partners, but do not need so.
D. immunoglobulin (Ig)
For differentiating that the HF-related agents of HF-associated protein comprises specific binding in the function equivalent of the immune globulin bletilla immunoglobulin (Ig) of HF-related polypeptide.Term " immunoglobulin (Ig) " and " antibody " are used interchangeably and use with its widest meaning in this article.Therefore, " immunoglobulin (Ig) " and " antibody " comprises intact monoclonal antibodies, polyclonal antibody, the multi-specificity antibody that formed by least two complete antibodies (such as, bispecific antibody) and antibody fragment, as long as they show the biologically active of expection.In one embodiment, patient's immunoglobulin (Ig) comprises at least one human constant domain.In another embodiment, HF-related agents immunoglobulin (Ig) comprise show with human constant domain at least about 90% to 95% sequence identity but keep the constant domain of mankind's effector function.IgH F-related agents or its function equivalent can be that the mankind, chimeric, humanized, mouse, CDR-grafting, phage display, bacteria display, yeast display, transgenic mice produces, mutagenesis with randomized.
I. general antibody
Term " antibody " and " immunoglobulin (Ig) " comprise can the antibody assembled completely of conjugated antigen and antibody fragment (such as, Fab ', F ' (ab) 2, Fv, single-chain antibody, the little antibody of bivalent), comprise recombinant antibodies and antibody fragment.Preferably, immunoglobulin (Ig) or antibody be chimeric, the mankind's or humanized.
The defined epitope of the variable domains identification isogeneic of heavy chain and light chain or be incorporated into the defined epitope of isogeneic.Term " epi-position " refer to immunoglobulin (Ig) variable end combine antigen on specific binding site or antigenic determinant.Epi-position can be linear, that is, be made up of the amino acid residue sequence found in former HF-correlated series.Epi-position also can be conformation, thus makes immunoglobulin (Ig) be identified in the 3-D structure found in folding HF-correlation molecule.Epi-position also can be combination that is linear and conforma-tional element.In addition, the carbohydrate portions as the molecule of the tumor cells expression by target azimuth also can be epi-position.
If: 1) immunoglobulin (Ig) shows the threshold level of binding activities, and/or 2) immunoglobulin (Ig) do not carry out cross reaction significantly with known related polypeptide molecule, so thinks that described immunoglobulin (Ig) is " specific binding ".Those of ordinary skill in the art easily can measure the binding affinity of immunoglobulin (Ig), such as, analyze (Scatchard, Ann.NYAcad.Sci.51:660-672,1949) measure by Scatchard.In some embodiments, immunoglobulin (Ig) of the present invention is with higher than other albumen at least 10 3doubly, more preferably at least 104 times, more preferably at least 10 5doubly, even more preferably at least 10 6doubly be horizontally bound on HF-associated protein.
Ii. polyclonal antibody and monoclonal antibody
Immunoglobulin (Ig) of the present invention can be polyclonal or monoclonal, and can be produced by any method well known in the art.
Preferably, polyclonal antibody is injected related antigen and adjuvant by (im) in (ip) in repeatedly subcutaneous (sc), peritonaeum or muscle and produces in animal.It can be useful for related antigen being incorporated into albumen, and described albumen is immunogenic in the species treating immunity.In addition, the aggregating agent of such as alum and so on or other medicaments are applicable to strengthen immune response.
Term " monoclonal antibody " refers to the antibody obtained from the antibody population of basic homology.Monoclonal antibody is high degree of specificity, for single antigen site.In addition, different from the preparation of the polyclonal antibody of the different antibodies typically comprised for different determinant, each monoclonal antibody is for the single determinant of antigen.
Except its specificity, monoclonal antibody be also advantageous in that they can be synthesized and do not polluted by other immunoglobulin (Ig)s.Such as, monoclonal antibody produces by hybridoma method or by recombinant DNA method.Monoclonal antibody HF-related agents also can be separated from phage antibody library.
Iii. chimeric antibody and humanized antibody
Can from a species (such as rodent) according to variable region, constant region can from the second species (such as people) this definition, and HF-related polypeptide binding domain-immunoglobulin or antibody can be " being fitted together to ".
" humanization " form of non-human HF-associated protein binding antibody is chimeric antibody, and described chimeric antibody contains the minimum sequence from non-human immunoglobulin.In most cases, humanized antibody is the human immunoglobulin (receptor antibody) that the residue of wherein acceptor hypervariable region is replaced by the residue (donor antibody) with the non-human species hypervariable region of the specificity of expectation, compatibility and ability, and described non-human species is mouse, rat, rabbit or non-human primates such as.Under certain situation, skeleton district (FR) residue of human immunoglobulin is replaced by corresponding non-human residues.In addition, humanized antibody can comprise the residue do not found in receptor antibody or donor antibody.
Usually, humanized antibody can comprise at least one substantially whole variable domains, and typically, humanized antibody can comprise two substantially whole variable domains, wherein, all or basic all high become ring correspond to non-human immunoglobulin Gao Bianhuan and all or basic all FR be the FR of human immunoglobulin sequence.In one embodiment, humanized antibody comprise show with the sequence identity of acceptor (non-human) FR (such as, mouse FR) be at least 65% humanization FR.Humanized antibody also can comprise constant region for immunoglobulin (Fc) (especially human immunoglobulin) at least partially.
This area has described carries out humanized method to non-human antibody.Preferably, humanized antibody contains one or more amino acid residues introduced from nonhuman origin.These non-human amino acid residues are commonly referred to " importing " residue, and these non-human amino acid residues typically take from " importing " variable domains.The corresponding sequence of humanization substantially by replacing human antibodies with hypervariable region sequence realizes.Therefore, such humanized antibody is chimeric, wherein, replaces the region being substantially less than complete human variable-domain by the corresponding sequence in non-human species.In fact, typically, humanized antibody is human antibodies, in this human antibodies, and some some hypervariable region residues and may be that some FR residues are replaced by the residue in the similar site of rodent antibodies.The selection being ready to use in the human variable-domain's (light chain and heavy chain) preparing humanized antibody is very important to reduction antigenicity.
Additive method generally comprises antagonist receptor variable region skeleton and gives donor CDR binding affinity.A kind of method comprises grafting simultaneously and optimizes the binding affinity of variable region binding fragment.Other method relates to the binding affinity optimizing antibody variable region.
Iv. antibody fragment
" antibody fragment " comprises a part for complete antibody, preferably its antigen binding domain or variable region.The example of antibody fragment comprises Fab, Fab ', F (ab ') 2, Fv fragment, the little antibody of bivalent, linear antibodies, single-chain antibody molecules and the multi-specificity antibody that formed by antibody fragment.
Antagonist carries out papain digestion and produces two identical Fabs, and be called " Fab " fragment, each fragment has single antigen binding site, and " Fc " fragment of remnants.Fab fragment also comprises the constant region of light chain and first constant region (CHI) of heavy chain.
The F of pepsin generation containing two antigen binding sites (ab ') 2fragment and this fragment still can with antigen crosslinking.Fab ' fragment and Fab fragment difference are that the heavy chain CHI domain carboxyl terminal of Fab ' fragment with the addition of several residue, and described residue comprises one or more halfcystines of antibody hinge region.Fab '-SH is the Fab ' title in this article that the cysteine residues of wherein constant domain has at least one free sulfydryl.F (ab ') 2originally antibody fragment produces as paired Fab ' fragment, containing hinge cysteine between described paired Fab ' fragment.Other chemical couplings of antibody fragment are known in the art.
" Fv " is the minimum antibody fragment comprising intact antigen identification and antigen binding site.This region is made up of with the dimer that Non-covalent binding mode is formed closely a heavy-chain variable domains and a light variable domains.In this configuration, three hypervariable regions of each variable domains interact with the antigen binding site limiting VH-VL dimer interface.Antibody antigen binding specificity is jointly given in six hypervariable regions.But even single variable domains (or only comprising the half of Fv of three hypervariable regions to antigen-specific) also has the ability to identify and conjugated antigen, although affinity is lower than whole binding site.
" scFv " or " scFv " antibody fragment comprises VH and the VL domain of antibody, and wherein, these domains are present in single polypeptied chain.Fv polypeptide can comprise the polypeptide linker between VH and VL domain further, and described polypeptide linker can make scFV form the expected structure of antigen combination.See PLUCKTHUN, 113THEPHARMACOLOGYOFMONOCLONALANTIBODIES269-315 (Rosenburg and Moore chief editor 1994).In addition see international publication WO93/16185, No. the 5th, 587,458, United States Patent (USP) and the 5th, 571, No. 894.
Multiple technologies are developed in order to produce antibody fragment.Traditionally, these fragments are obtained by proteolytic digestion complete antibody.But these fragments can directly be produced by recombinant host cell now.
V. combine and mark
Anti-HF-associated protein antibody can their " exposed " forms or uncombined form administration, maybe can have other medicament combined with them.
Such as antibody can be the form that can mark with detecting.Antibody can be marked by using radioactive isotope, affinity labeling (such as biotin, Avidin etc.), enzyme mark (such as horseradish peroxidase, alkaline phosphatase etc.), fluorescence labeling (such as FIFC or rhodamine etc.), paramagnetic atom etc. with detecting.The process realizing such mark is known in the art.
Vi. bispecific antibody
Bispecific antibody of the present invention is the little antibody fragment containing two antigen binding sites.Each fragment comprises heavy-chain variable domains (VH), and described heavy-chain variable domains (VH) is connected to the light variable domains (VL) in same polypeptied chain (VH-VL).By using length too short so that cannot make the connector of two domain pairings on same chain, domain is forced to match with the complementary domain of another chain and produce two antigen binding sites.
The method preparing bispecific antibody is known in the art.The traditional mode of production of total length bispecific antibody is based on the right coexpression of two heavy chain immunoglobulin-light chains, and wherein two chains have different specificitys.
In other method, the antibody variable territory (antibody-antigene binding site) with the binding specificity of expection can be blended in immunoglobulin constant domains sequence.Particularly, variable domains and heavy chain immunoglobulin constant domain merge, and described heavy chain constant domain comprises at least part of hinge area, CH2 district and CH3 district.In one embodiment, fusion comprises the first CH (CHI), because it contains light chain in conjunction with required site.Encode immunoglobulin heavy merges and the polynucleotide of light chain immunoglobulin (if necessary) can to insert in independent expression vector also cotransfection in suitable HOST ORGANISMS.When three polypeptied chains of the inequality proportion used in construct provide optimum output, in the mutual ratio of three polypeptide fragments of said method in adjustment embodiment, provide the dirigibility of height.But when at least two polypeptied chains cause high yield with equal rates's expression, or when described speed does not have special meaning, the coded sequence inserting two or whole three polypeptied chains in an expression vector is possible.
Bispecific antibody also uses leucine zipper and scFv (sFv) dimer to produce.
E. the diagnosis for the treatment of of fibrosis, prognosis and assessment
Therefore, on the other hand, the invention provides the Fibrotic method of use HF-related polypeptide diagnosis and prognostic described herein.In concrete non-limiting embodiment, the method detecting the HF-related polypeptide in cell or serum/plasma, promote the diagnosis of fiberization seriousness in Fibrotic diagnosis and patient in patient, promote the determination of patient's prognosis, measure patient to Fibrotic neurological susceptibility and assessment patient to the response for the treatment of (such as, by providing the measurement to result for the treatment of (such as, among chemotherapy regimen or afterwards assess tumor burden)) in useful.These methods can comprise the level detecting HF-related polypeptide in patient biological sample (such as, serum/plasma or doubtful or be about to the fibrosed tissue that produces or cell).Detection method of the present invention can be implemented in external or body on the cell be separated or in whole tissues or body fluid (such as, blood, blood plasma, serum, urine, etc.).In one embodiment, HF-related polypeptide can be used to detect and assesses fiberization.These biomarkers can be applicable to show Fibrotic any disease, such as liver fibrosis, kidney fibrosis, cardiac fibrosis, fibrosis of skin, pancreatic fibrosis etc., but more preferably, described fiberization is liver fibrosis.
F. HF-related polypeptide is detected
The invention provides the method detecting HF-related polypeptide in serum or blood plasma.Any one in various known method all can be used for detecting, include but not limited to: the functional detection using the immunoassays of the specific antibody of the polypeptide of coding and the polypeptide to coding, described use coding polypeptide specific antibody immunoassays such as, by enzyme linked immunosorbent assay (ELISA) (ELISA), radiommunoassay (RIA), protein spots trace, Western blot, turbidimetry, scattered light urbidmetry etc.; The functional test example of the described polypeptide to coding is as biologically active.HF-related polypeptide also can use non-antibody approach to come quantitatively, and described non-antibody approach carries out multiple-reaction monitoring such as, but not limited to, use mass spectrum.
After reading instructions of the present invention, it is evident that for those of ordinary skills, detection method as herein described and additive method can be easy to change.Within the scope that such change is expected in the present invention.Such as, in superincumbent detection scheme, the probe used in detection can be fixed on solid support, and test sample book and fixing probes touch.The combination of test sample book and probe can detect subsequently in every way, such as, is incorporated into the detectable label of test sample book by detecting, thus is conducive to detecting the probe complex of test sample book-fixing.
The present invention further provides the existence of HF-related polypeptide in the detection of specific antibody biological specimen using HF-related polypeptide and/or measure the method for HF-related polypeptide level.Particularly, the method detecting the existence of HF-related polypeptide in biological specimen can comprise following steps: sample is contacted with monoclonal antibody and detects the combination of antibody and HF-related polypeptide in sample.More specifically, antibody can use compound to mark, thus produces detectable signal, and described compound includes but not limited to: radioactive label, enzyme, chromophore and fluorophor.
Time compared with suitable contrast, detect that the specific antibody of HF-related polypeptide or the specific binding of its function equivalent are the symbols that HF-related polypeptide is present in sample.Suitable contrast comprises the known sample not containing HF-related polypeptide and the sample contacted with the antibody (such as, anti-idiotype antibody) of the non-specific polypeptide for encoding.The interactional various method of detection specificity antibody-antigene known in the art also can be used for following method, includes but not limited to, standard immunoassay Histological method, immunoprecipitation, EIA enzyme immunoassay and radiommunoassay.Generally speaking, specific antibody will can be marked directly or indirectly with detecting.Direct mark comprises radioactive isotope; The detectable enzyme of product (such as, luciferase, 3-galactosidase, etc.); Fluorescence labeling (such as, fluorescein isothiocynate, rhodamine, phycoerythrin, etc.); The metal (such as, being connected to the 112Eu of antibody or other members of lanthanide series by the metal chelating groups of such as EDTA and so on) of emitting fluorescence; Chemiluminescence compound (such as, luminol, different luminol, acridinium salt, etc.); Bioluminescent compound (such as, fluorescein, aequorin (green fluorescent protein), etc.).Antibody can connect (coupling) in insoluble holder, such as polystyrene board or pearl.The specificity two that non-immediate mark comprises the specific antibody (" specificity primary antibodie ") of the polypeptide of coding resists the member with specific binding partners, wherein, described two anti-are labeled as described above, and the member of described specific binding partners such as, biotin-avidin, etc.Biological specimen can contact and on the solid support being fixed on such as nitrocellulose and so on or carrier, described solid support or carrier can fixed cell, cell granulations or soluble proteins.Described holder can wash with suitable damping fluid subsequently, then contacts with the specificity primary antibodie that can mark with detecting.Detection method is known in the art and the signal launched according to detectable label is selected detection method.Detect general by carrying out contrasting realizing with appropriate control and appropriate criteria.
G. kit
Detection method can be used as the part of kit and provides.Therefore, the present invention is also provided for detecting the existence of HF-related polypeptide in biological specimen and/or detecting the kit of HF-related polypeptide level.Use the step of these kits can be undertaken by clinical labororatory, experimental laboratory, doctor or individual.Kit of the present invention is for detecting the HF-related polypeptide of differential expression in fibrotic processes.This kit may be provided in additional component useful in step, includes, but not limited to buffering agent, developping agent, mark, reaction surface, detection method, check sample, standard, instructions and explain information.
embodiment
The differentially expressed protein set up when the blood plasma relatively from healthy individuals and the blood plasma from liver cirrhosis patient
Time compared with healthy individuals, inventor of the present invention finds differential expression in the human plasma sample of the liver cirrhosis patient that multiple protein is brought out at HCV-.This discovery is compared these plasma samples by using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and is realized, described two-dimensional polyacrylamide gel electrophoresis be a kind of on gel-type vehicle two-way protein isolate obtain the technology of discrete protein spot.Use the 2D-PAGE of wide region pH3 to pH10 in international publication WO/2008/031051, previously use the biomarker differentiated in the serum of liver fibrosis.In the present invention, the discriminating of fiberization biomarker is different from international publication WO/2008/031051, because employ close limit pH3 to pH5.6.This pH scope is selected to be because it is outside the scope of the main isoform of the highest plasma/serum albumen (albumin, IgG, siderophillin) of three wealth of species.This is the discovery first this pH3 to pH5.6 scope being used for biomarker.
The discovery of new biomarker in disease is by the restriction spanning the dynamic protein concentration scope of ten orders of magnitude in serum and plasma.Therefore, high-abundance proteins (especially albumin, immunoglobulin (Ig) and siderophillin) limits the detection of low-abundance protein.In order to overcome this problem found in biomarker, a lot of people attempted based on antibody and based on the initial gross separation strategy of dye affinity high-abundance proteins exhausted from sample before electrophoresis thus to improve the performance of low-abundance protein.But, immuno-precipitation is expensive, because need lot of antibodies to exhaust these abundant albumen, and dyestuff affine method efficiency is lower and specificity is lower, along with a large amount of non-albuminous unnecessary removal, and therefore may eliminate potential biomarker in Proteomic analysis.Unlike the present invention use a large amount of albumen (2mg), due to the recovery problem in high-abundance proteins removing process and the loss in concentration process, multiple sample after exhaustion with the similar very challenging property of high-level albumen application of sample.
Although there is the problem of high-abundance proteins in blood plasma, in wide region pH3 to pH10,2D-PAGE in international publication WO/2008/031051, has been used to be successfully authenticated several candidate new biomarkers of liver fibrosis.In the application, the biomarker of this group liver fibrosis is increased by using the 2D-PAGE of close limit pH3 to pH5.6, this scope outside the main isoform scope of albumin, immunoglobulin (Ig) and siderophillin, thus makes many four times of the protein ratio international publication WO/2008/031051 of application of sample.This makes the performance of low abundance unique point be improved and separation is improved.Use this pH scope to achieve serum acidic protein group and blood plasma acidic protein group and be able to remarkable improvement based on the separation of gel, and sightless low abundance liver fibrosis biomarker in the international publication WO/2008/031051 using wide region pH3 to pH10 is differentiated.
Embodiment 1
Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE)
In order to differentiate the biomarker of the liver scarring that HCV-brings out, analyze the plasma sample (often organizing 6 individualities) of the liver cirrhosis patient that the plasma sample of healthy individuals and HCV-bring out in based on the protein science research of 2D-PAGE.Whole plasma sample is collected in P100 pipe (BD, Oxford, UK).These P100 blood plasma pipes are selected to be because different from other blood collection tubes, they contain proprietary protein stabiliser, described proprietary protein stabiliser dissolves at once when blood is collected, described proprietary protein stabiliser improves recovery and the preservation of albumen, makes them be applicable to the discovery of proteome analysis and biomarker.Different from the international publication WO/2008/031051 analyzed from the serum of patients with liver fibrosis in various degree, the present invention only concentrates the albumen inquiring into differential expression between the sample from healthy individuals and the sample from liver cirrhosis patient.The method is adopted to be that this shows that in this research, analyze these interstages can not produce other candidate's fiberization biomarker any because all differences expressing protein previously differentiated in mild fibrosis and moderate fibrosis is also found in cirrhosis.In gel, use the nonlinear gradient of pH3 to pH5.6 by charge separation first 2 milligrams of plasma proteinss, then pass through molecular weight (size) separation second to the SDS-PAGE gradient of middle use 9% to 16% (w/v).As Gangadharan etc., (2007) at Clin.Chem., 53, carry out electrophoresis, fluorescent dye and gel image scanning like that described in 1792.
Embodiment 2
Differential image is analyzed and is differentiated with albumen
By computer-aided image analysis, between normal plasma sample with cirrhosis plasma sample, compare the two-dimensional array of the spot of generation.The scan image of whole 2D-PAGE gel is by such as Gangadharan etc., and (2007), Clin.Chem., the computer-aided image analysis described in 53,1792 is analyzed.The differential expression change being more than or equal to 2 times of differences is considered to significant.As Gangadharan etc., (2007), Clin.Chem., 53, described in 1792, altogether the unique point of 57 differential expressions cut, carry out mass spectrophotometry with Trypsin Induced.
Embodiment 3
Differentiate the human plasma biomarker of the liver fibrosis that cirrhosis causes
Differential image analysis shows the primary evidence of potential blood plasma biomarker.See Fig. 1 to Fig. 4.This analysis display fat metastasis suppressor albumen, zinc-α-2-glycoprotein, the β hoptoglobin at pH5.46 to pH5.49 place, hoptoglobin associated protein, apoC-III, apo E, C4b-associated proteins β chain, paraoxonase/Arylesterase activity 1, retinol-binding proteins, first albumin, α-2-HS-glycoprotein, corticosteroid-binding globulin, the expression of rich leucine α-2-glycoprotein and fibrinogen γ chain decreases in cirrhosis serum, and full complement C3dg, immunoglobulin (Ig) J chain, sex hormone binding globulin, 14-3-3 albumen ζ/δ, adiponectin and the antitryptic expression of α-1-add.Also observe the posttranslational modification of glycoprotein hemopexin.
Fiberization biomarker described in international publication WO/2008/031051 is also differentiated: CD5 antigen sample albumen and Ig α/κ chain increase; Alpha-1-antichymotrypsin analogues, CLU, Complement C4, inter-α-trypsin inhibitor heavy chain H4 and transthyretin reduce.With regard to inter-α-trypsin inhibitor heavy chain H4, confirm that the uncracked form of this albumen decreases by the analysis of mass spectrum to peptide sequence, and previously in international publication WO/2008/031051, to have only had the crack fragment of 35kDa and 70kDa to be identified as be reduce.
All above-mentioned albumen is top score albumen by mass spectral analyses.In gel character point, identify other lower score albumen, although can not be excluded, described lower score albumen can not be comparatively the reason producing differential expression change.The lower score albumen identified is that Ig λ chain and apolipoprotein AI increase, and albumin, AMBP, factor, pigment epidermal derived factors and serum amyloid sample P-component reduce.
Embodiment 4
The measurement of these novel proteins in serum/plasma will contribute to the reliable diagnosis of fiberization and cirrhosis, can reduce the needs of liver biopsy.These measurements can use the immunoassays of the antibody of these albumen of target and realize.
Find that a fragment of Complement C_3 increases and observes the 39kDa place of the isoelectric point of described fragment about pI4.9 on 2D-PAGE gel in cirrhosis.It is 955 to 1201 by the Amino Acid Range of this fragment of Mass Spectrometric Identification.The Amino Acid Range of Complement C_3 dg be 955 to 1303 and its theoretical molecular conform to viewed gel character point with isoelectric point (39kDa with pI5), prove that the Complement C_3 in this unique point is Complement C_3 dg.Known Complement C_3 dg has at amino acid/11 010 to 1013 place can by the thioesters site of fibrinolysin (enzyme that fiber hydrolization is relevant) cracking.Known fibrinolysin reduces in fiberization, and this is consistent with the level that the uncracked Complement C_3 dg observed in cirrhosis increases.In international publication WO/2008/031051, find that a fragment of Complement C_3 reduces in fiberization and cirrhosis, observe described fragment at 49kDapI6.9 place, and the amino acid of Mass Spectrometric Identification shows that it is the α chain of the Complement C_3 before thioesters site, prove that the cracking to Complement C_3 of fibrinolysin mediation in fiberization reduces (Gangadharan etc., (2007), Clin.Chem., 53,1792).Therefore in the present invention, the antibody of the plasmin cleavage sites (namely overlapping with thioesters site, amino acid/11 010 to 1013 place) of targeting complement C3 will contribute to the degree determining cracking.Seem at present not for the commercially antibody of Complement C_3 thioesters site areas.Antibody for cracking zone can help the increase level determining uncracked Complement C_3 in liver scarring more reliably.
Known total binding globin reduce in fiberization and at present and other albumen together use with diagnosing liver fibrosis (see international publication WO0216949).In the present invention, the 2D-PAGE unique point of the β hoptoglobin found containing pH5.46 to pH5.49 place reduces and seems more more reliable than total binding globin in cirrhosis.Known hoptoglobin contains four potential glycosylation sites, and described glycosylation site is all on its β chain.In plasma/serum, β hoptoglobin is glycosylated usually.When by 2D-PAGE separated plasma/serum, find that β hoptoglobin is the equally spaced unique point of a row between pH4.7 and pH5.8, described unique point can be shown as minimizing in liver scarring.In the present invention, the 2D-PAGE gel character point of hoptoglobin at pH5.46 to pH5.49 place reduces and other unique points of β hoptoglobin between seeming than pH4.7 and pH5.8 are more reliable in cirrhosis.
Embodiment 5
The fibrosis grade of each in new biomarker can be formulated.Measure the mean concentration of these biomarkers in the serum of liver fibrosis different phase.Use the classified estimation liver fibrosis of 0 to 6 at present clinically, wherein 0 represent non-fiberization, the fiberization interstage that 1-5 representative increases from light to moderate/serious seriousness, 6 is cirrhosis (Ishak, (1995), JHepatol, 22,696).By determining the concentration range of the new biomarker in these seven stages, the similar points-scoring system from 0 to 6 can be specified.The result that the score value of all new biomarker is added will provide more reliable fibrosis index, but not detect single biomarker.
Embodiment 6
Immunoassays
The invention provides for assessment of Fibrotic kit, described kit detects HF-related polypeptide.This realizes by using the antibody being incorporated into HF-related polypeptide.These antibody can be used for carrying out immunoassays, such as enzyme linked immunosorbent assay (ELISA) (ELISA), radioimmunoassay, protein spots trace, Western blot, turbidimetry, scattered light urbidmetry etc.HF-related polypeptide also can use non-antibody approach to come quantitatively, and described non-antibody approach carries out multiple-reaction monitoring such as, but not limited to, use mass spectrum.
With regard to ELISA, detection can be carried out in 96 orifice plates.A kind of selection uses noncompetitive unit point in conjunction with ELISA.In this detection, prepare the antigen (in this embodiment, referring to new biomarker and serum sample) of concentration known with bicarbonate buffer, described antigen is added 96 orifice plates and at 4 DEG C night incubation.Then use the solution (PBS-T) of phosphate buffered saline (PBS) (PBS) and tween to wash plate three times, then close by the PBS solution containing bovine serum albumin(BSA).After hatching 1 hour at 37 DEG C, add the primary antibodie for antigen (in this embodiment, feeling the pulse with the finger-tip mark biomarker), hatch plate at 37 DEG C and wash three times with PBS-T again in 1 hour.Then add two anti-(animal origins for primary antibodie) that horseradish peroxidase combines, and at 37 DEG C, hatch plate 1 hour, then wash three times with PBS-T.Finally, add such as 2 to every hole, the peroxidase substrate of 2 '-azine-bis-(3-ethyl benzo thiazole phenanthroline-6-sulfonic acid) and so on, reads the absorbance at 405nm place in microplate reader after 30 minutes.
Alternatively, sandwich ELISA can be used.In such detection, a kind of antibody is incorporated into the bottom of plate hole.Add antigen (referring to biomarker protein in this situation), and by the unconjugated product of washing removing.Then another antibody be labeled being incorporated into this antigen is added.The amount of another antibody combined is carried out quantitatively (usually passing through than colourity).Except new biomarker, inventor of the present invention proposes hoptoglobin and measures by ELISA.Hoptoglobin level (liver functional test in conjunction with clinician measures) in serum will set up the more reliable score being used for liver fibrosis.
Although foregoing relates to specific preferred implementation, it is to be appreciated that the present invention is not limited thereto.Those of ordinary skill in the art will make various change to disclosed embodiment and these changes will within the scope of the present invention.
The full content of all public publications quoted in this instructions, patented claim and patent is incorporated to herein by reference.

Claims (3)

1. fat transfer inhibitor albumen purposes in the kit for the preparation of the liver fibrosis detected and in the assessment serum of human patients or plasma sample, wherein compares the liver fibrosis for detecting and assess in serum in described human patients or plasma sample with the control level of described fat transfer inhibitor albumen by the level of fat transfer inhibitor albumen in the serum of described human patients or plasma sample.
2. fat transfer inhibitor albumen purposes in the kit of the severity rankings for the preparation of the liver fibrosis in the serum of human patients or plasma sample, wherein compares the severity rankings for the liver fibrosis in the serum of described human patients or plasma sample with the control level of described fat transfer inhibitor albumen by the level of fat transfer inhibitor albumen in the serum of described human patients or plasma sample.
3. the purposes of fat transfer inhibitor albumen in the kit of the prognosis for the preparation of the liver fibrosis measured in the serum of human patients or plasma sample, wherein compares the prognosis of the liver fibrosis in serum for measuring described human patients or plasma sample with the control level of described fat transfer inhibitor albumen by the level of fat transfer inhibitor albumen in the serum of described human patients or plasma sample.
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